This application focuses on homeostatic coupling of intracellular copper (Cu) and iron (Fe) levels to gene expression in the yeast Saccharomyces cerevisiae. Metalloregulation of Cu uptake genes is controlled by the Mac1 transcriptional activator. Metalloregulation of Fe uptake genes is controlled by two related activators, Aft1 and Aft2. These three factors are transcriptional activators in metal-deficient cells, but specifically inhibited in metal-replete cells. The major objectives are to elucidate the mechanisms of Cu ion sensing by Mac1 and Fe ion sensing by Aft1 and Aft2. Many interconnections exist between Fe and Cu homeostasis in yeast. In the past grant cycle we provided evidence suggestive that Mac1 senses Cu ions within the yeast nucleus through direct binding of Cu(I) ions by Mac1. Cu(I) binding appears to induce an intramolecular interaction between the N-terminal DNA binding domain and C-terminal transactivation domain. Studies are proposed to verify direct sensing of Cu and to map the intramolecular interface that inhibits both DNA binding and transactivation. An important goal is to identify other yeast proteins important in either Cu shuttling to the nucleus or Cu inhibition of Mac1. Fe-homeostasis in yeast is regulated by Aft1 and Aft2. The mechanism of Fe inhibition of Aft1 and Aft2 function is unknown. The significance of these studies is that S. cerevisiae is the best model eukaryotic system to understand the mechanism of Fe sensing by a transcription regulator. We propose to determine whether Fe sensing occurs through direct Fe binding, Fe-mediated post-translational modification or through another protein? Aft1 and Aft2 exhibit limited functional redundancy in yeast. We propose to determine whether each exhibits a distinct physiological function. Mac1 and Aftl/Aft2 are excellent model eukaryotic systems to study nutritional control of gone expression. Information gleaned on the mechanism of Cu- and Fe-regulation may be applicable to other species.